Characterization of an antibody to the integrin β3 subunit (GP IIIa) from a patient with neonatal thrombocytopenia and an inherited deficiency of GP IIb-IIIa complexes in platelets (Glanzmann's thrombasthenia)

Patient A.F. is a 28-year-old poly transfused woman with an inherited bleeding disorder, Olanzmann's thrombasthenia. An abnormal platelet function is linked to severe decreases in the platelet content of the integrins GP IIb and GP IIIa. In 1987 the patient gave birth to a child with severe anemia and thrombocytopenia. Serological tests revealed the presence of anti-platelet antibody together with an anti-Rhesus D. Western blotting identified a major antibody that reacted with a protein of 90-95 kDa present in platelets and endothelial cells. This was identified as the β3 integrin subunit (GP IIIa). Antibody-binding required intact disulfides, while controlled digestion with proteases showed the determinant(s) to be retained within chymotrypsin- (50, 63 kDa) and Staphylococcus aureus V8 protease-derived (25-38 kDa) fragments of GP IIIa. Direct binding assays performed in the presence of monoclonal antibodies specific for different epitopes on GP IIb-IIIa complexes confirmed that the epitope was exposed on intact platelets and revealed a specific inhibition of A.F. IgG binding by the monoclonal antibody, AP-3. Other tests confirmed that the antibody reacted independently of the Pl^A or Pen polymorphisms carried by GP IIIa. IgG purified from A.F. plasma by adsorption and elution from paraformaldehyde-fixed normal platelets or electrophoretically separated GP IIIa was an inhibitor of ADP-induced platelet aggregation. Unexpectedly, Western blotting showed trace amounts of abnormally migrating GP IIIa in A.F. platelets, which retained an ability to react with her antibody. This suggests that the patient has formed an autoantibody reactive with an active site of the β3 integrin subunit and linked to the development of neonatal thrombocytopenia.